Our investigation of the mechanism of the reaction that forms the O-alkyl ether bond in glycerolipids continued during the past year. Preparations of alkyl-DHAP (dihydroxyacetone-P) synthase, the enzyme that catalyzes the formation of the ether bond, used for these experiments were purified 1000-fold from Ehrlich ascites cell microsomes. In addition to catalyzing the forward reaction (the conversion of acyl-DHAP to alkyl-DHAP), alkyl-DHAP synthase also catalyzes several intermediate steps including the exchange of the acyl moiety of acyl-DHAP with free fatty acids; such an exchange is consistent with the ping-pong mechanism that we had previously proposed. Recent data obtained with both [unreadable]3[unreadable]H-\and [unreadable]18[unreadable]O-labeled palmitoyl-DHAP have demonstrated that the purified enzyme does not hydrolyze the acyl group of this substrate in the absence of the cosubstrate hexadecanol. Earlier studies by our laboratory revealed that acyl-CoA reductase (the enzyme that forms the long-chain fatty alcohol precursor of the ether bond) was tenaciously embedded in the microsomal membrane matrix. Our attempts to solubilize acyl-CoA reductase for purification were unsuccessful, and therefore our current knowledge about the conversion of acyl-CoA to fatty alcohols via a transient aldehyde intermediate and the regulation of this important enzyme is based on experiments done with relatively complex systems. The regulation of acyl-CoA reductase by a soluble protein factor in mouse preputial glands was originally reported by us two years ago. Since the level of soluble protein stimulator of acyl-CoA reductase increased during development of the gland in male mice, we became interested in knowing whether testosterone was the physiological factor responsible for regulating the activity of acyl-CoA reductase in the preputial gland during development of the mouse in puberty. Daily injections of testosterone into castrated mice increases the activity of acyl-CoA reductase in preputial glands to the same level as that of normal controls, whereas the activity of acyl-CoA reductase is barely detectable in castrated mice not receiving testosterone. The half-life of acyl-CoA reductase calculated from the decay curve of the enzyme induction is 69 hrs. These results indicate that testosterone is important in controlling the activity of acyl-CoA reductase and thereby influences the levels of fatty alcohols available for ether lipid synthesis. (B)